Abstract Obesity and associated metabolic diseases including type 2 diabetes is a current epidemic in the US and worldwide. There are no current safe anti-obesity drugs available and bariatric surgery, in combination with dietary and physical activity regimens, is considered the best option to treat obese patients. Thus, there is an urgent medical need to identify new therapeutic targets and develop new and safer drugs to treat obesity. Defects in energy expenditure in response to diet or lower temperatures cause obesity. A major site of energy expenditure is the brown or beige adipose tissue that contains thermogenic mitochondria equipped to uncouple respiration and produce heat. The presence in humans of these thermogenic adipocytes opens up the possibility to activate them and protect against obesity. In the previous funded period we have found that deficiency of the protein Clk2, a kinase downstream of feeding signals and insulin, in adipose tissue decreases energy expenditure and exacerbates body weight upon high fat diet feeding. Clk2-deficient brown adipocytes exhibit a failure in thermogenic function associated with low levels of Uncoupling protein 1. However, the regulatory components and mechanisms of how the feeding-regulated Clk2 kinase activity affects cold- and diet-induced thermogenic activity in brown and beige adipose cells and control whole body energy expenditure are unknown. The major goal of this grant renewal is to identify and analyze the molecular and regulatory mechanism whereby the protein kinase Clk2 activates thermogenic function in brown/beige adipose tissue in response to cold and diet and increases energy expenditure to protect against obesity and diabetes. The research strategy is focused on three central aims: 1) Molecular and functional analysis of how Clk2 kinase controls thermogenic gene expression programs in brown/beige adipose cells (Specific Aim 1), 2) Cellular, metabolic and bioenergetic analysis mediated by Clk2 kinase action in adipose cells and fat tissues ex vivo (Specific Aim 2) and, 3) In vivo metabolic and energetic analysis driven by Clk2 kinase in cold- and diet- induced thermogenesis (Specific Aim 3). The outcomes from these studies will identify novel molecular mechanisms and regulatory components by which brown and beige adipocytes control rates of energy expenditure and protect against diet-induced obesity. Since insufficient energy expenditure is a hallmark of obesity and associated pathologies, our studies may translate into potential therapies.